Cancer vaccines and vaccination methods

ABSTRACT

Methods and compositions for treating cancers (e.g., neural cancers) by dendritic cell vaccination are provided herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority from U.S. ProvisionalPatent Application Ser. No. 60/827,260, filed on Sep. 28, 2006, theentire contents of which are incorporated herein by reference.

TECHNICAL FIELD

The invention relates to methods and compositions for the treatment ofcancers, such as neural cancers.

BACKGROUND

Brainstem gliomas are a heterogeneous group of tumors that can bedistinguished by age of onset, clinical and radiological presentation,and biological behavior. The diagnosis of a diffuse brainstem glioma isbased upon typical imaging, dispensing with the need for surgery in themajority of cases. Radiation therapy is the mainstay of treatment forchildren with diffuse brainstem gliomas. The role of chemotherapy forthese children is not clear, and it is, in general, employed in thecontext of an investigational study. Less than 10% of children withdiffuse brainstem gliomas survive 2 years. In contrast to childhoodbrainstem gliomas, adult brainstem gliomas are rare and poorlyunderstood. Mean age at onset is 34 years. The main presenting symptomsare gait disturbance (61%), headache (44%), weakness of the limbs (42%)and diplopia (40%). The diagnosis of a brainstem glioma is uniformlylethal. Glioblastoma is the most common and most malignant primary braintumor. Survival with surgery, radiation, and chemotherapy ranges from 12to 15 months.

The potential therapy is immunotherapy, which is a form of cancertreatment that activates the immune system to attack and eradicatecancer cells. Cytotoxic T lymphocytes (“CTL”) are critical to asuccessful antitumor immune response. T cells that attack cancer cellsrequire the presentation of tumor antigens to naïve T cells that undergoactivation, clonal expansion, and ultimately exert their cytolyticeffector function. Unfortunately, this mechanism is defective inpatients with malignant gliomas. Effective antigen presentation isessential to successful CTL effector function. Thus, the development ofa successful strategy to initiate presentation of tumor antigens to Tcells can be important to an immunotherapeutic strategy for malignantbrain tumors (Liu et al., Expert Rev. Vaccines, 5(2):233-247, 2006).

Various immunotherapies have been investigated for malignant glioma,including antibody- and cytokine-based therapies, cancer vaccines, andadoptive cellular therapies. However, such treatments for centralnervous system gliomas have not been discovered as quickly as therapiesfor more immunogenic tumors, e.g., melanoma. This is partly due to therelative lack of defined glioma-associated antigens that can be targetedby the immune system. In recent years, several tumor-associated antigens(“TAA”) have been identified and characterized for different cancers,including breast, colon, renal, and melanoma.

Some tumor-associated antigens have been identified for human gliomacells, including tyrosinase-related protein (TRP)-2 (a melanomadifferentiation antigen), Melanoma-associated Antigen-1 (MAGE-1), acancer/testis antigen, HER-2/neu (selectively overexpressed in tumors),interleukin-13 (IL-13) receptor α2, gp100 (a melanoma differentiationantigen), and Antigen isolated from Immunoselected Melanoma-2 (AIM-2), anovel tumor antigen (Liu et al., Oncogene, 24(33): 5226-5234, 2005; Liuet al., J. Immunother., 26(4): 301-312, 2003; Liu et al., J.Immunother., 27(3): 220-226, 2004; Liu et al., Canc. Res., 64:4980-4986, 2004).

With respect to these specific antigens, a vaccine consisting ofdendritic cells pulsed with MAGE-1 peptide has been used in melanomapatients to induce clinical and systemic tumor-specific responseswithout provoking major side effects. It has also been shown thatmelanoma patients immunized with a melanoma cell vaccine induce antibodyresponses to recombinant MAGE-1 antigen. In addition, several clinicaltrials have indicated that gp100 is a highly immunogenic antigen inmelanoma patients, and showed a strong correlation between T cellrecognition of the gp100 antigen and clinical responses. The HER-2oncogenic protein has been well-defined in the art, and a HER-2 specificvaccine has been tested in human clinical trials. Early results showedthe immunity elicited by the vaccine was durable even after vaccinationsended.

The immunogenicity and regulation of HER-2, gp100, and MAGE-1 inglioblastoma multiforme (“GBM”) have been investigated. Liu et al.(Canc. Res., 64: 4980-4986, 2004) describes that the majority of GBMsexpress these antigens and process the dominant epitopes. It was alsodetermined that CTLs recognize these antigens on GBMs, and thatrecognition is determined by both antigen expression and MHC expressionon the cell surfaces. These results showed that tumor antigen expressionin GBM cells correlates with tumor cell recognition by CTLs.

With respect to the antigen AIM-2, it has been shown that bothnon-spliced and spliced AIM-2 transcripts are expressed in many tumortypes. One particular melanoma-reactive T cell clone recognizes apeptide from non-spliced AIM-2, but not from spliced AIM-2. GBMs expressAIM-2—spliced and non-spliced forms—and process the dominant epitopefrom non-spliced AIM-2, allowing CTL recognition of peptides. Inaddition, AIM-2 CTL have been generated in certain patients byvaccination with dendritic cells pulsed with tumor lysates, and that theability of CTLs to recognize autologous tumor cells was increased bythese vaccinations.

TRP-2 is a naturally processed, immunogenic tumor antigen in mice andhumans. Vaccination with dendritic cells pulsed with TRP-2 has beenshown to generate TRP-2-specific CTLs and immunity against B16 melanomatumors, delay B16 tumor growth, and prolong mouse survival. It was alsodemonstrated that immunization with the human TRP-2 gene elicitedautoantibodies and autoreactive cytotoxic T cells. TRP-2-specificcytotoxic T cell activity has been detected in patients aftervaccination with dendritic cells pulsed with autologous tumor lysate. Ina dendritic cell-based phase I clinical trial, TRP-2 peptide-specificCTLs were induced in patients without observed side effects orautoimmune reactions. It has also been demonstrated that GBM cells frompostvaccination resections show lower TRP-2 expression and highersensitivity to chemotherapeutic drugs than autologous cell lines frompre-vaccination resections.

With the diagnosis of a brainstem glioma being uniformly lethal,glioblastoma as the most common and most malignant primary brain tumor,and survival with surgery, radiation, and chemotherapy only ranging from12 to 15 months, there exists a significant need in the art for thedevelopment of novel therapeutic measures.

SUMMARY

The invention is based, in part, on the discovery that immunizing gliomapatients with antigen presenting cells (APC) loaded with uniquecombinations of multiple tumor antigens induces therapeutic immuneresponses that can be used to treat these patients to providesignificantly increased survival. Accordingly, methods for inducingimmune responses in cancer patients (e.g., neural cancer patients, suchas glioma patients) against tumor antigens are provided herein. Themethods use as vaccines APC, such as dendritic cells (DC), that presentspecific combinations of multiple different tumor antigens. Alsoprovided are compositions that include the cells and the antigens.

Various embodiments provide for vaccines including epitopes of anycombination of four or more of the following antigens:tyrosinase-related protein (TRP)-2, Melanoma-associated Antigen-1(MAGE-1), HER-2, IL-13 receptor α2, gp100, and AIM-2. For example, thevaccines include epitopes (e.g., peptide fragments) of any four of theantigens, any five of the antigens, or all six of the antigens. In someembodiments, the vaccines include epitopes for additional tumorantigens.

Additional embodiments of the present invention provide for vaccinesloaded with one or more superagonist epitopes for some or all of thefollowing antigens: TRP-2, MAGE-1, HER-2, IL-13 receptor α2, gp100, andAIM-2. A “superagonist” or “superantigen” peptide is a peptide thatincludes one or more mutations (e.g., one, two, or three amino acidchanges, relative to a native sequence) and that elicits anantigen-specific immunological response that is more potent than aresponse elicited by a peptide having a native sequence. For example, asuperagonist peptide stimulates higher levels of IFN-γ release byantigen-specific T cells, as compared to T cells stimulated with thenative peptide. The increase in levels of IFN-γ release stimulated by asuperagonist peptide are higher than levels stimulated by a nativepeptide by a statistically significant amount. In some embodiments, asuperagonist stimulates IFN-γ levels that are at least 5%, 10%, 25%,50%, 100%, 200%, or 500% higher than elicited by the native peptide.

The vaccines of the present invention can be used to treat a cancer,e.g., a neural cancer. In particular embodiments, the vaccines can beused to treat gliomas. In other embodiments the vaccines can be used totreat glioblastoma multiforme (GBM). In other embodiments, the vaccinescan be used to treat astrocytomas. In various embodiments, the vaccinesare administered in an amount sufficient to induce an immune responseagainst the antigens (e.g., a T cell response).

The vaccines can include autologous dendritic cells. In alternativeembodiments, the vaccines can include allogeneic dendritic cells.Dendritic cells suitable for use in the vaccination methods disclosedherein can be isolated or obtained from any tissue in which such cellsare found, or can be otherwise cultured and provided. Dendritic cellscan be found in, for example, but in no way limited to, the bone marrow,peripheral blood mononuclear cells (PBMCs) of a mammal, or the spleen ofa mammal. Additionally, any suitable media that promote the growth ofdendritic cells can be used in accordance with the present invention,and can be readily ascertained by one skilled in the art.

The dendritic cells in the vaccines described herein can be pulsed withany or all of the following antigens (i.e., incubated for a sufficienttime to allow uptake and presentation of peptides of the antigens on MHCmolecules): TRP-2, MAGE-1, HER-2, IL-13 receptor α2, gp100, and AIM-2,or epitopes of these antigens (e.g., peptide epitopes 7-25 amino acidsin length). The epitopes are, for example, peptides 7 to 13 (e.g., 8 to10, e.g., 9) amino acids in length.

The dendritic cells present epitopes corresponding to the antigens at ahigher average density than epitopes present on dendritic cells exposedto a tumor lysate (e.g., a neural tumor lysate) (e.g., at a density thatis at least 5%, 10%, 25%, 50%, 100%, or 200% higher). The dendriticcells can acquire the antigens or portions thereof (e.g., peptideepitopes) by incubation with the antigens in vitro (e.g., wherein cellsacquire antigens by incubation with the combination of the antigenssimultaneously, or with a subset of antigens, e.g., in separate pools ofcells). In some embodiments, the dendritic cells are incubated with acomposition including the peptides, wherein the peptides are syntheticpeptides and/or were isolated or purified prior to incubation with thecells. In some embodiments, dendritic cells are engineered to expressthe peptides by recombinant means (e.g., by introduction of a nucleicacid that encodes the full length antigen or a portion thereof, e.g.,the peptide epitope).

In some embodiments, the synthetic peptides include a synthetic peptidehaving a dibasic motif (i.e., Arg-Arg, Lys-Lys, Arg-Lys, or Lys-Arg) atthe N-terminus and a dibasic motif at the C-terminus. In someembodiments, the synthetic peptides include a HER2 peptide including oneof the following amino acid sequences: RRILHNGAYSLRR (SEQ ID NO:1) orRRKIFGSLAFLRR (SEQ ID NO:2).

The dendritic cells can include a peptide including an amino acidsequence corresponding to an epitope of TRP-2, MAGE-1, HER-2, IL-13receptor α2, gp100, and AIM-2, described herein. For example, thedendritic cells include at least one of the following sequences:RSDSGQQARY (SEQ ID NO:3) from AIM-2; EADPTGHSY (SEQ ID NO:4) fromMAGE-1; SVYDFFVWL (SEQ ID NO:5) from TRP-2; ITDQVPFSV (SEQ ID NO:6) fromgp100; KIFGSLAFL (SEQ ID NO:7) from HER-2; and WLPFGFILI (SEQ ID NO:8)from IL-13 receptor α2. In some embodiments, the peptide is amidated atthe C-terminus.

In alternative embodiments, the dendritic cells in the vaccines arepulsed with any or all of superagonist epitopes of some or all of theaforementioned antigens. The superagonist antigens have certain aminoacid substitutions that generate a more potent immune response than thenatural epitopes. In some embodiments, the dendritic cells are pulsedwith a peptide epitope including one or both of the followingsuperagonist peptide sequences: YMDQVPYSV (SEQ ID NO:65) from gp100; orFMANVAIPHL (SEQ ID NO:68) from HER-2. In some embodiments, the dendriticcells are pulsed with a peptide epitope including one of the followingpeptide sequences: FLDQVPYSV (SEQ ID NO:63) from gp100; ILDQVPFSV (SEQID NO:66) from gp100; IMDQVPFSV (SEQ ID NO:67) from gp100, FMHNVPIPYL(SEQ ID NO:69) from HER-2; or FYANVPSPHL (SEQ ID NO:70) from HER-2. Insome embodiments, the peptide is amidated at the C-terminus.Superagonist peptides can be used in combination with any of thepeptides described herein.

In some embodiments, the dendritic cells include more than one peptideepitope for a given antigen, e.g., wherein the dendritic cells comprisetwo, three, four, or more peptide epitopes from AIM-2, and/or two,three, four, or more peptide epitopes from MAGE-1, and so forth.

Other embodiments of the present invention provide for methods oftreating cancers (e.g., neural cancers, e.g., gliomas) using theinventive vaccines. In one embodiment, the method of treating gliomascomprises administering a vaccine as described herein to a patient.Other embodiments provide for methods of treating cancers such ascarcinomas, or brain metastatic cancers.

The vaccines can be administered one or more times to a patient toimpart beneficial results. The vaccines can be administered prior orpost surgical resection of the tumor. One skilled in the art will beable to determine the appropriate timing for administering the vaccine.The timing of the first and/or subsequent dose(s) of the vaccine candepend on a variety of factors, including, but not limited to apatient's health, stability, age, and weight. The vaccine can beadministered at any appropriate time interval; for example, includingbut not limited to, once per week, once every two weeks, once everythree weeks, once per month. In one embodiment, the vaccine can beadministered indefinitely. In one embodiment, the vaccine can beadministered three times in two week intervals. Appropriate dosages ofthe vaccines also depends on a variety of factors, including, but notlimited to, a patient's health, stability, age, and weight. In oneembodiment, the vaccine includes from about 10⁵ to about 10⁹ tumorantigen-pulsed dendritic cells. In another embodiment, the vaccineincludes about 10⁷ tumor antigen-pulsed dendritic cells.

In some embodiments, the methods of treating cancers include identifyinga patient whose tumor expresses one or more of TRP-2, MAGE-1, HER-2,IL-13 receptor α2, gp100, and AIM-2, prior to the treatment. Forexample, a method can include evaluating whether a tumor in a gliomapatient expresses HER-2, and, if the tumor expresses HER-2,administering the vaccine to the patient. Patients whose tumors arepositive for other tumor antigens can also be identified and selectedfor treatment.

The vaccines can be administered in conjunction with other therapeutictreatments; for example, chemotherapy and/or radiation. In someembodiments, the inventive vaccines are administered by injection (i.e.,intravenous, intraarterial, etc.). In other embodiments, the inventivevaccines are administered directly into or in close proximity of thetumor. In other embodiments, the inventive vaccines are administereddirectly into or in close proximity of the site of the resected tumor.

In other embodiments, methods of producing the inventive vaccines areprovided. In some embodiments, the vaccines are made by obtainingdendritic cells from a subject and loading the dendritic cells with theantigens. The dendritic cells can be autologous or allogeneic.

In some embodiments, a method of producing the vaccine includesobtaining bone marrow derived mononuclear cells from a subject,culturing the mononuclear cells in vitro under conditions in whichmononuclear cells become adherent to a culture vessel, selecting asubset of the mononuclear cells including adherent cells, culturing thesubset of cells in the presence of one or more cytokines (e.g., GM-CSF,IL-4, TNF-α) under conditions in which the cells differentiate intoantigen presenting cells, culturing the adherent cells in the presenceof synthetic peptides, the peptides including amino acid sequencescorresponding to epitopes of at least four of the following sixantigens: TRP-2, MAGE-1, HER-2, IL-13 receptor α2, gp100, and AIM2,under conditions in which the cells present the peptides on majorhistocompatibility class I molecules, thereby preparing a cell vaccine.In some embodiments, the bone marrow derived cells are obtained from apatient with a cancer (e.g., a neural cancer, e.g., glioma), and thecell vaccine is prepared to treat the patient.

In some embodiments, the synthetic peptides include a synthetic peptidehaving a dibasic motif (i.e., Arg-Arg, Lys-Lys, Arg-Lys, or Lys-Arg) atthe N-terminus and a dibasic motif at the C-terminus. In someembodiments, the synthetic peptides include a HER2 peptide including oneof the following amino acid sequences: RRILHNGAYSLRR (SEQ ID NO:1) orRRKIFGSLAFLRR (SEQ ID NO:2).

In another aspect, the invention features a peptide fragments of TRP-2,MAGE-1, IL-13 receptor α2, gp100, and AIM2, modified to include dibasicmotifs at the N-terminus and C-terminus (e.g., a peptide having one ofthe following amino acid sequences: RRRSDSGQQARYRR (SEQ ID NO:9);RREADPTGHSYRR (SEQ ID NO:10); RRSVYDFFVWLRR (SEQ ID NO:11);RRITDQVPFSVRR (SEQ ID NO:12); and RRWLPFGFILIRR (SEQ ID NO:13).Combinations of the peptides, and compositions including the peptidesare also provided.

This invention also provides immunogenic compositions that include, orencode the combinations of antigens described herein, and methods ofusing the compositions. For example, preparations of HER-2, AIM-2,MAGE-1, TRP-2, IL-13 receptor α2, and gp100 peptides, for use as cancervaccines (e.g., peptide vaccines, or nucleic acids encoding thepeptides) are provided. The invention also provides immunogeniccompositions that include a superagonist peptide, e.g., a superagonistpeptide epitope corresponding to one or more of HER-2, AIM-2, MAGE-1,TRP-2, IL-13 receptor α2, and gp100.

“Beneficial results” can include, but are in no way limited to,lessening or alleviating the severity of the disease condition,preventing the disease condition from worsening, curing the diseasecondition, and prolonging a patient's life or life expectancy.

“Cancer” and “cancerous” refer to or describe the physiologicalcondition in mammals that is typically characterized by unregulated cellgrowth. Examples of neural cancers include cancers of the brain andspinal cord such as gliomas, glioblastomas, glioblastoma multiforme(GBM), oligodendrogliomas, primitive neuroectodermal tumors, low, midand high grade astrocytomas, ependymomas (e.g., myxopapillary ependymomapapillary ependymoma, subependymoma, anaplastic ependymoma),oligodendrogliomas, medulloblastomas, meningiomas, pituitary adenomas,neuroblastomas, and craniopharyngiomas. GBM, glioblastomas,astrocytomas, ependymomas, and oligodendrogliomas are types of gliomas.

“Conditions” and “disease conditions,” as used herein can include, butare in no way limited to any form of neoplastic cell growth andproliferation, whether malignant or benign, pre-cancerous and cancerouscells and tissues; in particular, gliomas, glioblastomas, glioblastomamultiforme (GBM), oligodendrogliomas, primitive neuroectodermal tumors,low, mid and high grade astrocytomas, ependymomas (e.g., myxopapillaryependymoma papillary ependymoma, subependymoma, anaplastic ependymoma),oligodendrogliomas, medulloblastomas, meningiomas, pituitary adenomas,neuroblastomas, and craniopharyngiomas.

“Mammal” as used herein refers to any member of the class Mammalia,including, without limitation, humans and nonhuman primates such aschimpanzees and other apes and monkey species; farm animals such ascattle, sheep, pigs, goats, and horses; domestic mammals such as dogsand cats; laboratory animals including rodents such as mice, rats, andguinea pigs, and the like. The term does not denote a particular age orsex. Thus, adult and newborn subjects, as well as fetuses, whether maleor female, are intended to be included within the scope of this term.The terms “patient” and “subject” are used interchangeably herein, andcover mammals including humans.

“Pathology” of cancer includes all phenomena that compromise thewell-being of the patient. This includes, without limitation, abnormalor uncontrollable cell growth, metastasis, interference with the normalfunctioning of neighboring cells, release of cytokines or othersecretory products at abnormal levels, suppression or aggravation ofinflammatory or immunological response, neoplasia, premalignancy,malignancy, invasion of surrounding or distant tissues or organs, suchas lymph nodes, etc.

“Treatment” and “treating,” as used herein refer to both therapeutictreatment and prophylactic or preventative measures, wherein the objectis to inhibit or slow down (lessen) the targeted disorder (e.g., cancer,e.g., glioma) or symptom of the disorder, or to improve a symptom, evenif the treatment is partial or ultimately unsuccessful. Those in need oftreatment include those already diagnosed with the disorder as well asthose prone or predisposed to contract the disorder or those in whom thedisorder is to be prevented. For example, in tumor (e.g., cancer)treatment, a therapeutic agent can directly decrease the pathology oftumor cells, or render the tumor cells more susceptible to treatment byother therapeutic agents or by the subject's own immune system.

“Tumor,” as used herein refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues.

A “dendritic cell” or “DC” is an antigen presenting cell (APC) thattypically expresses high levels of MHC molecules and co-stimulatorymolecules, and lacks expression of (or has low expression of) markersspecific for granulocytes, NK cells, B lymphocytes, and T lymphocytes,but can vary depending on the source of the dendritic cell. DCs are ableto initiate antigen specific primary T lymphocyte responses in vitro andin vivo, and direct a strong mixed leukocyte reaction (MLR) compared toperipheral blood leukocytes, splenocytes, B cells and monocytes.Generally, DCs ingest antigen by phagocytosis or pinocytosis, degradeit, present fragments of the antigen at their surface and secretecytokines.

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Singleton et al., Dictionary ofMicrobiology and Molecular Biology 3rd ed., J. Wiley & Sons (New York,N.Y. 2001); March, Advanced Organic Chemistry Reactions, Mechanisms andStructure 5th ed., J. Wiley & Sons (New York, N.Y. 2001); Sambrook andRussel, Molecular Cloning: A Laboratory Manual 3rd ed., Cold SpringHarbor Laboratory Press (Cold Spring Harbor, N.Y. 2001); and Lutz etal., Handbook of Dendritic Cells: Biology, Diseases and Therapies, J.Wiley & Sons (New York, N.Y. 2006), provide one skilled in the art witha general guide to many of the terms used in the present application.Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and notintended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, the drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting levels of epitope-specific T cell responsesin mice following immunization IV with mouse lysozyme-M (ML-M), a mouselysozyme peptide (p19-30), and a mouse lysozyme peptide modified withdibasic residues (18R31R), as measured by cell proliferation in vitro. Tcells were isolated from lymph nodes of the animals (3 animals pergroup) and response to each immunogen was measured.

FIGS. 2A and 2B are bar graphs that show IFN-γ levels after in vitroinduction of CTL in patient-derived PBMCs with dendritic cells pulsedwith gp100 (2M) or superagonist gp100 peptides in accordance with anembodiment of the present invention in two separate patients.

FIGS. 3A and 3B are bar graphs that show IFN-γ levels after in vitroinduction of CTL in patient-derived PBMCs with native or superagonistHer-2 peptides in accordance with an embodiment of the present inventionin two different patients.

FIG. 4 is a bar graph that shows IFN-γ levels produced by CTL after invitro induction of the CTL in patient-derived PBMCs with native (no. 52)or altered Her-2 peptides (nos. 19, 32, and 41), as measured by ELISA.CTL were cocultured with Her-2 positive, gp100 positive cell lines.

FIG. 5 is a bar graph that shows numbers of IFN-γ positive cells, asmeasured by ELISPOT analysis of CTL generated against Her-2 peptide nos.19, 32, 41, and 52. For the ELISPOT assays, CTL were incubated with T2cells pulsed with a HER-2 native peptide.

FIG. 6 is a bar graph that shows IFN-γ levels produced by CTL after invitro induction of the CTL in patient-derived PBMCs with a native (no.2M) or altered gp100 peptides (nos. 8, 22, 38, 62, and 63), as measuredby ELISA. CTL were cocultured with Her-2 positive, gp100 positive celllines.

FIG. 7 is a is a bar graph that shows numbers of IFN-γ positive cells,as measured by ELISPOT analysis of CTL generated against gp100 peptidenos. 2M, 8, 22, 38, 62, and 63. For the ELISPOT assays, CTL wereincubated with T2 cells pulsed with a gp100 native peptide.

DETAILED DESCRIPTION

The invention provides, inter alia, methods and compositions fortreating gliomas by administering cells presenting unique combinationsof tumor antigens. Vaccination with dendritic cells or GM-CSF secretingcells is safe and elicits a cytotoxic T cell response associated withmemory T cells with dendritic cells and naïve T cells with GM-CSF (Yu,J. S., Wheeler, C. J., Zeltzer, P. M., et al., Cancer Res, 61: 842-847,2001). The combinations of antigens described herein elicit therapeutic,tumor-specific immune responses. The combinations of antigens describedherein stimulate a more heterogeneous immune response than would beelicited with a single antigen, and thus are particularly beneficial fortargeting tumors. For example, a tumor may evolve such that expressionof a given tumor antigen is turned off. Thus, an immune response againstmultiple tumor antigens is more likely to provide effective therapy inthis context, and can provide significant therapeutic benefits forvarious patient populations. The present compositions and methodsfeature combinations including epitopes from four, five, or six of thefollowing: TRP-2, MAGE-1, HER-2, IL-13 receptor α2, gp100, and AIM-2.Tables 1 and 2 lists amino acid sequences of these antigens and peptideepitopes of the antigens.

Tumor Antigens

AIM-2

AIM-2 is expressed in a variety of tumor types, includingneuroectodermal tumors, and breast, ovarian and colon carcinomas. Table1 provides an amino acid sequence of human AIM-2 (also available inGenBank under accession no. AAD51813.1, GI: 5802881).

The following is an exemplary sequence of an AIM-2 HLA epitope:RSDSGQQARY (SEQ ID NO:3) (also shown in Table 2, below). This epitope isencoded by an alternative open reading frame (see Harada et al., J.Immunother., 24(4):323-333, 2001).

GP100

Gp100 is a glycoprotein preferentially expressed in melanocytes. Table 1provides an amino acid sequence of human gp100 (also available inGenBank under accession no. NP_(—)008859.1, GI: 5902084). Table 2 listsexemplary HLA epitopes from gp100.

HER-2

HER-2 (also known as HER-2/neu, and c-erbB2) is a transmembraneglycoprotein with tyrosine kinase activity. HER-2 is overexpressed in avariety of tumor types.

Table 1 provides an amino acid sequence of human HER-2 (also availablein GenBank under accession no. NP_(—)004439.2, GI: 54792096). Table 2lists exemplary HLA epitopes from HER-2.

IL-13 Receptor α2

IL-13 receptor α2 is a non-signaling component of the multimeric IL-13receptor. An exemplary human IL-13 receptor α2 amino acid sequence isshown in Table 1 (also available in Genbank under acc. no.NP_(—)000631.1, GI: 10834992).

The following is an exemplary sequence of an IL-13 receptor α2 HLAepitope, corresponding to amino acids 345-354 of the above sequence:WLPFGFILI (SEQ ID NO:8) (also shown in Table 2).

MAGE-1

MAGE-1 is a cancer/testis antigen originally identified in melanoma.

Table 1 provides an amino acid sequence of human MAGE-1 (also availablein GenBank under accession no. NP_(—)004979.3, GI: 148276977). Table 2lists exemplary MAGE-1 HLA peptide epitopes.

TRP-2

TRP-2 is a dopachrome tautomerase involved in melanogenesis (Aroca etal., Biochim Biophys Acta., 1035(3):266-75, 1990). Human TRP-2 shares84% identity with murine TRP-2 (Yokoyama et al., Biochim. Biophys.Acta., 1217:317-321, 1994). TRP-2 has five isoforms generated byalternative poly(A) site usage or alternative splicing, including theisoforms designated as TRP-2-6b, TRP-2-INT2, TRP-2-LT, and TRP-2-8b. SeeLiu et al., J. Immunother., 26(4):301-312, 2003; Pisarra et al., J.Invest. Dermatol., 115:48-56, 2000; Khong and Rosenberg, J. Immunol.,168:951-956, 2002; and Lupetti et al., J. Exp. Med., 188:1005-1016,1998. Epitopes of each of these isoforms are useful for the vaccines andmethods disclosed herein.

Table 1 provides a sequence of human TRP-2 which has 519 amino acids(also available in GenBank under accession no. NP_(—)001913.2,GI:6041667). The amino acid sequence of another human TRP-2 isoform thathas 552 amino acids is available in Genbank under acc. no. ABI73976.1,GI:114384149. Table 2 lists exemplary TRP-2 HLA epitopes.

TABLE 1 Tumor antigen Amino acid sequence AIM-2MVVLGMQTEEGHCIMLRGLAPSLGGTQVICKVVGLPSSIGFNTSSHLLFPATLQGAPTHFPCRWRQGGSTDNPPA (SEQ ID NO: 14) gp100MDLVLKRCLLHLAVIGALLAVGATKVPRNQDWLGVSRQLRTKAWNRQLYPEWTEAQRLDCWRGGQVSLKVSNDGPTLIGANASFSIALNFPGSQKVLPDGQVIWVNNTIINGSQVWGGQPVYPQETDDACIFPDGGPCPSGSWSQKRSFVYVWKTWGQYWQVLGGPVSGLSIGTGRAMLGTHTMEVTVYHRRGSRSYVPLAHSSSAFTITDQVPFSVSVSQLRALDGGNKHFLRNQPLTFALQLHDPSGYLAEADLSYTWDFGDSSGTLISRALVVTHTYLEPGPVTAQVVLQAAIPLTSCGSSPVPGTTDGHRPTAEAPNTTAGQVPTTEVVGTTPGQAPTAEPSGTTSVQVPTTEVISTAPVQMPTAESTGMTPEKVPVSEVMGTTLAEMSTPEATGMTPAEVSIVVLSGTTAAQVTTTEWVETTARELPIPEPEGPDASSIMSTESITGSLGPLLDGTATLRLVKRQVPLDCVLYRYGSFSVTLDIVQGIESAEILQAVPSGEGDAFELTVSCQGGLPKEACMEISSPGCQPPAQRLCQPVLPSPACQLVLHQILKGGSGTYCLNVSLADTNSLAVVSTQLIMPGQEAGLGQVPLIVGILLVLMAVVLASLIYRRRLMKQDFSVPQLPHSSSHWLRLPRIFCSCPIGENSPLLSGQQV (SEQ ID NO: 15) HER-2MELAALCRWGLLLALLPPGAASTQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYYNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTSIISAVVGILLVVVLGVVFGILIKRRQQKIRKYTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYNIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLGPASPLDSTFYRSLLEDDDMGDLVDAEEYLVPQQGFFCPDPAPGAGGMVHHRHRSSSTRSGGGDLTLGLEPSEEEAPRSPLAPSEGAGSDVFDGDLGMGAAKGLQSLPTHDPSPLQRYSEDPTVPLPSETDGYVAPLTCSPQPEYVNQPDVRPQPPSPREGPLPAARPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLTPQGGAAPQPHPPPAFSPAFDNLYYWDQDPPERGAPPSTFKGTPTAENPEYLGLDVPV (SEQID NO: 16) IL-13MAFVCLAIGCLYTFLISTTFGCTSSSDTEIKVNPPQDFEIVDPGYLGYLYLQWQPPLSLDHFKECTVEYEreceptorLKYRNIGSETWKTIITKNLHYKDGFDLNKGIEAKIHTLLPWQCTNGSEVQSSWAETTYWISPQGIPETKVα2QDMDCVYYNWQYLLCSWKPGIGVLLDTNYNLFYWYEGLDHALQCVDYIKADGQNIGCRFPYLEASDYKDFYICVNGSSENKPIRSSYFTFQLQNIVKPLPPVYLTFTRESSCEIKLKWSIPLGPIPARCFDYEIEIREDDTTLVTATVENETYTLKTTNETRQLCFVVRSKVNIYCSDDGIWSEWSDKQCWEGEDLSKKTLLRFWLPFGFILILVIFVTGLLLRKPNTYPKMIPEFFCDT (SEQ ID NO: 17) MAGE-MSLEQRSLHCKPEEALEAQQEALGLVCVQAATSSSSPLVLGTLEEVPTAGSTDPPQSPQGASAFPTTINF 1TRQRQPSEGSSSREEEGPSTSCILESLFRAVITKKVADLVGFLLLKYRAREPVTKAEMLESVIKNYKHCFPEIFGKASESLQLVFGIDVKEADPTGHSYVLVTCLGLSYDGLLGDNQIMPKTGFLIIVLVMIAMEGGHAPEEEIWEELSVMEVYDGREHSAYGEPRKLLTQDLVQEKYLEYRQVPDSDPARYEFLWGPRALAETSYVKVLEYVIKVSARVRFFFPSLREAALREEEEGV (SEQ ID NO: 18) TRP-2MSPLWWGFLLSCLGCKILPGAQGQFPRVCMTVDSLVNKECCPRLGAESANVCGSQQGRGQCTEVRADTRPWSGPYILRNQDDRELWPRKFFHRTCKCTGNFAGYNCGDCKFGWTGPNCERKKPPVIRQNIHSLSPQEREQFLGALDLAKKRVHPDYVITTQHWLGLLGPNGTQPQFANCSVYDFFVWLHYYSVRDTLLGPGRPYRAIDFSHQGPAFVTWHRYHLLCLERDLQRLIGNESFALPYWNFATGRNECDVCTDQLFGAARPDDPTLISRNSRFSSWETVCDSLDDYNHLVTLCNGTYEGLLRRNQMGRNSMKLPTLKDIRDCLSLQKFDNPPFFQNSTFSFRNALEGFDKADGTLDSQVMSLHNLVHSFLNGTNALPHSAANDPIFVVLHSFTDAIFDEWMKRFNPPADAWPQELAPIGHNRMYNMVPFFPPVTNEELFLTSDQLGYSYAIDLPVSVEETPGWPTTLLVVMGTLVALVGLFVLLAFLQYRRLRKGYTPLMETHLSSKRYTEEA (SEQ ID NO: 19)

TABLE 2 Tumor Antigen Peptides Position in Tumor antigen sequencePeptide sequence AIM-2 RSDSGQQARY (SEQ ID NO: 3) gp100  71-78 SNDGPTLI(SEQ ID NO: 20) gp100 154-162 KTWGQYWQV (SEQ ID NO: 21) gp100 209-217ITDQVPFSV (SEQ ID NO: 6) gp100 280-288 YLEPGPVTA (SEQ ID NO: 22) gp100613-622 SLIYRRRLMK (SEQ ID NO: 23) gp100 614-622 LIYRRRLMK (SEQ ID NO:24) gp100 619-627 RLMKQDFSV (SEQ ID NO: 25) gp100 639-647 RLPRIFCSC (SEQID NO: 26) gp100 476-485 VLYRYGSFSV (SEQ ID NO: 27) HER-2   5-13ALCRWGLLL (SEQ ID NO: 28) HER-2   8-16 RWGLLLALL (SEQ ID NO: 29) HER-2 63-71 TYLPTNASL (SEQ ID NO: 30) HER-2 106-114 QLFEDNYAL (SEQ ID NO: 31)HER-2 369-377 KIFGSLALFL (SEQ ID NO: 7) HER-2 435-443 ILHNGAYSL (SEQ IDNO: 32) HER-2 654-662 IISAVVGIL (SEQ ID NO: 33) HER-2 665-673 VVLGVVFGI(SEQ ID NO: 34) HER-2 689-697 RLLQETELV (SEQ ID NO: 35) HER-2 754-762VLRENTSPK (SEQ ID NO: 36) HER-2 773-782 VMAGVGSPYV (SEQ ID NO: 37) HER-2780-788 PYVSRLLGI (SEQ ID NO: 38) HER-2 789-797 CLTSTVQLV (SEQ ID NO:39) HER-2 799-807 QLMPYGCLL (SEQ ID NO: 40) HER-2 835-842 YLEDVRLV (SEQID NO: 41) HER-2 851-859 VLVKSPNHV (SEQ ID NO: 42) HER-2 883-899KVPIKWMALESILRRRF (SEQ ID NO: 43) HER-2 952-961 YMIMVKCWMI (SEQ ID NO:44) HER-2 971-979 ELVSEFSRM (SEQ ID NO: 45) IL-13 receptor 345-354WLPFGFIILI α2 (SEQ ID NO: 8) MAGE-1 102-112 ITKKVADLVGF (SEQ ID NO: 46)MAGE-1 135-143 NYKHCFPEI (SEQ ID NO: 47) MAGE-1 160-169 KEADPTGHSY (SEQID NO: 48) MAGE-1 161-169 EADPTGHSY (SEQ ID NO: 4) MAGE-1 230-238SAYGEPRKL (SEQ ID NO: 49) MAGE-1 278-286 KVLEYVIKV (SEQ ID NO: 50) TRP-2180-188 SVYDFFVWL (SEQ ID NO: 5) TRP-2 197-205 LLGPGRPYR (SEQ ID NO: 51)TRP-2 222-231 EVISCKLIKR (TRP-2- (SEQ ID NO: 52) int2 isoform) TRP-2288-296 SLDDYNHLV (SEQ ID NO: 53) TRP-2 360-368 TLDSQVMSL (SEQ ID NO:54) TRP-2 387-395 ANDPIFVVL (SEQ ID NO: 55) TRP-2 399-407 LLYNATTNI (SEQID NO: 56) TRP-2 403-411 ATTNILEHY (SEQ ID NO: 57) TRP-2 402-411NATTNILEHV (SEQ ID NO: 58) TRP-2 455-463 YAIDLPVSV (SEQ ID NO: 59)

Antigenic peptides useful for loading DCs for vaccination are peptidesthat stimulate a T cell mediated immune response (e.g., a cytotoxic Tcell response) by presentation to T cells on MHC molecules. Therefore,useful peptide epitopes of TRP-2, MAGE-1, gp100, AIM-2, IL-3 receptorα2, and HER-2, include portions of the amino acid sequences that bind toMHC molecules and are presented to T cells. Peptides that bind to MHCclass I molecules are generally 8-10 amino acids in length. Peptidesthat bind to MHC class II molecules are generally 13 amino acids orlonger (e.g., 13-17 amino acids long).

T cell epitopes can be identified by a number of different methods.Naturally processed MHC epitopes can be identified by massspectrophotometric analysis of peptides eluted from antigen-loaded APC(e.g., APC that have taken up antigen, or that have been engineered toproduce the protein intracellularly). After incubation at 37° C., cellsare lysed in detergent and the MHC protein is purified (e.g., byaffinity chromatography). Treatment of the purified MHC with a suitablechemical medium (e.g., under acidic conditions, e.g., by boiling in 10%acetic acid, as described in Sanchez et al., 94(9): 4626-4630, 1997)results in the elution of peptides from the MHC. This pool of peptidesis separated and the profile compared with peptides from control APCtreated in the same way. The peaks unique to the protein expressing/fedcells are analyzed (for example by mass spectrometry) and the peptidefragments identified. This protocol identifies peptides generated from aparticular antigen by antigen processing, and provides a straightforwardmeans of isolating these antigens.

Alternatively, epitopes are identified by screening a synthetic libraryof peptides which overlap and span the length of the antigen in an invitro assay. For example, peptides which are 9 amino acids in length andwhich overlap by 5 amino acids may be used. The peptides are tested inan antigen presentation system that includes antigen presenting cellsand T cells. T cell activation in the presence of APCs presenting thepeptide can be measured (e.g., by measuring T cell proliferation orcytokine production) and compared to controls, to determine whether aparticular epitope is recognized by the T cells.

The peptides can be modified to increase immunogenicity. For example,addition of dibasic amino acid residues (e.g., Arg-Arg, Arg-Lys,Lys-Arg, or Lys-Lys) to the N- and C-termini of peptides can render thepeptides more potent immunogens.

The peptides can also include internal mutations that render them“superantigens” or “superagonists” for T cell stimulation. Superantigenpeptides can be generated by screening T cells with a positionalscanning synthetic peptide combinatorial library (PS-CSL) as describedin Pinilla et al. Biotechniques, 13(6):901-5, 1992; Borras et al., J.Immunol. Methods, 267(1):79-97, 2002; U.S. Publication No. 2004/0072246;and Lustgarten et al., J. Immun. 176:1796-1805, 2006. In someembodiments, a superagonist peptide is a peptide shown in Table 2,above, with one, two or three amino acid substitutions which render thepeptide a more potent immunogen.

Antigenic peptides can be obtained by chemical synthesis using acommercially available automated peptide synthesizer. Chemicallysynthesized peptides can be precipitated and further purified, forexample by high performance liquid chromatography (HPLC). Alternatively,the peptides can be obtained by recombinant methods using host cell andvector expression systems. “Synthetic peptides” includes peptidesobtained by chemical synthesis in vitro as well as peptides obtained byrecombinant expression. When tumor antigen peptides are obtainedsynthetically, they can be incubated with dendritic cells in higherconcentrations (e.g., higher concentrations than would be present in atumor antigen cell lysates, which includes an abundance of peptides fromnon-immunogenic, normal cellular proteins). This permits higher levelsof MHC-mediated presentation of the tumor antigen peptide of interestand induction of a more potent and specific immune response, and oneless likely to cause undesirable autoimmune reactivity against healthynon-cancerous cells.

Preparation of Antigen Presenting Cells

Antigen presenting cells (APC), such as dendritic cells (DC), suitablefor administration to subjects (e.g., glioma patients) may be isolatedor obtained from any tissue in which such cells are found, or may beotherwise cultured and provided. APC (e.g., DC) may be found, by way ofexample, in the bone marrow or PBMCs of a mammal, in the spleen of amammal or in the skin of a mammal (i.e., Langerhan's cells, whichpossess certain qualities similar to that of DC, may be found in theskin). For instance, bone marrow may be harvested from a mammal andcultured in a medium that promotes the growth of DC. GM-CSF, IL-4 and/orother cytokines (e.g., TNF-α), growth factors and supplements may beincluded in this medium. After a suitable amount of time in culture inmedium containing appropriate cytokines (e.g., suitable to expand anddifferentiate the DCs into mature DCs, e.g., 4, 6, 8, 10, 12, or 14days), clusters of DC cultured in the presence of antigens of interest(e.g., in the presence of peptide epitopes of AIM-2, gp100, HER-2,MAGE-1, and TRP-2, or a combination of at least five of these peptides)and harvested for use in a cancer vaccine using standard techniques.Antigens (e.g., isolated or purified peptides, or synthetic peptides)can be added to cultures at a concentration of 1 μg/ml-50 μg/ml perantigen, e.g., 2, 5, 10, 20, 30, or 40 μg/ml per antigen.

In one exemplary method of preparing APC, APC are isolated from asubject (e.g., a human) according to the following exemplary procedure.Mononuclear cells are isolated from blood using leukapheresis (e.g.,using a COBE Spectra Apheresis System). The mononuclear cells areallowed to become adherent by incubation in tissue culture flasks for 2hours at 37° C. Nonadherent cells are removed by washing. Adherent cellsare cultured in medium supplemented with granulocyte macrophage colonystimulating factor (GM-CSF) (800 units/ml, clinical grade, Immunex,Seattle, Wash.) and interleukin-4 (IL-4) (500 units/ml, R&D Systems,Minneapolis, Minn.) for five days. On day five, TNF-α is added to theculture medium for another 3-4 days. On day 8 or 9, cells are harvestedand washed, and incubated with peptide antigens for 16-20 hours on atissue rotator. Peptide antigens are added to the cultures at aconcentration of ˜10 μg/ml (per antigen).

Various other methods may be used to isolate the APCs, as would berecognized by one of skill in the art. DCs occur in low numbers in alltissues in which they reside, making isolation and enrichment of DCs arequirement. Any of a number of procedures entailing repetitive densitygradient separation, fluorescence activated cell sorting techniques,positive selection, negative selection, or a combination thereof areroutinely used to obtain enriched populations of isolated DCs. Guidanceon such methods for isolating DCs can be found in O'Doherty, U. et al.,J. Exp. Med., 178: 1067-1078, 1993; Young and Steinman, J. Exp. Med.,171: 1315-1332, 1990; Freudenthal and Steinman, Proc. Nat. Acad. Sci.USA, 57: 7698-7702, 1990; Macatonia et al., Immunol., 67: 285-289, 1989;Markowicz and Engleman, J. Clin. Invest., 85: 955-961, 1990;Mehta-Damani et al., J. Immunol., 153: 996-1003, 1994; and Thomas etal., J. Immunol., 151: 6840-6852, 1993. One method for isolating DCsfrom human peripheral blood is described in U.S. Pat. No. 5,643,786.

The dendritic cells prepared according to methods described hereinpresent epitopes corresponding to the antigens at a higher averagedensity than epitopes present on dendritic cells exposed to a tumorlysate (e.g., a neural tumor lysate). The relative density of one ormore antigens on antigen presenting cells can be determined by bothindirect and direct means. Primary immune response of naïve animals areroughly proportional to antigen density of antigen presenting cells(Bullock et al., J. Immunol., 170:1822-1829, 2003). Relative antigendensity between two populations of antigen presenting cells cantherefore be estimated by immunizing an animal with each population,isolating B or T cells, and monitoring the specific immune responseagainst the specific antigen by, e.g., tetramer assays, ELISPOT, orquantitative PCR.

Relative antigen density can also be measured directly. In one method,the antigen presenting cells are stained with an antibody that bindsspecifically to the MHC-antigen complex, and the cells are then analyzedto determine the relative amount of antibody binding to each cell (see,e.g., Gonzalez et al., Proc. Natl. Acad. Sci. USA, 102:4824-4829, 2005).Exemplary methods to analyze antibody binding include flow cytometry andfluorescence activated cell sorting. The results of the analysis can bereported e.g., as the proportion of cells that are positive for stainingfor an individual MHC-antigen complex or the average relative amount ofstaining per cell. In some embodiments, a histogram of relative amountof staining per cell can be created.

In some embodiments, antigen density can be measured directly by directanalysis of the peptides bound to MHC, e.g., by mass spectrometry (see,e.g., Purcell and Gorman, Mol. Cell. Proteomics, 3:193-208, 2004).Typically, MHC-bound peptides are isolated by one of several methods. Inone method, cell lysates of antigen presenting cells are analyzed, oftenfollowing ultrafiltration to enrich for small peptides (see, e.g., Falket al., J. Exp. Med., 174:425-434, 1991; Rotzxhke et al., Nature,348:252-254, 1990). In another method, MHC-bound peptides are isolateddirectly from the cell surface, e.g., by acid elution (see, e.g.,Storkus et al., J. Immunother., 14:94-103, 1993; Storkus et al., J.Immunol., 151:3719-27, 1993). In another method, MHC-peptide complexesare immunoaffinity purified from antigen presenting cell lysates, andthe MHC-bound peptides are then eluted by acid treatment (see, e.g.,Falk et al., Nature, 351:290-296). Following isolation of MHC-boundpeptides, the peptides are then analyzed by mass spectrometry, oftenfollowing a separation step (e.g., liquid chromatography, capillary gelelectrophoresis, or two-dimensional gel electrophoresis). The individualpeptide antigens can be both identified and quantified using massspectrometry to determine the relative average proportion of eachantigen in a population of antigen presenting cells. In some methods,the relative amounts of a peptide in two populations of antigenpresenting cells are compared using stable isotope labeling of onepopulation, followed by mass spectrometry (see Lemmel et al., Nat.Biotechnol., 22:450-454, 2004).

Administration of Antigen Presenting Cells

The APC-based cancer vaccine may be delivered to a patient or testanimal by any suitable delivery route, which can include injection,infusion, inoculation, direct surgical delivery, or any combinationthereof. In some embodiments, the cancer vaccine is administered to ahuman in the deltoid region or axillary region. For example, the vaccineis administered into the axillary region as an intradermal injection. Inother embodiments, the vaccine is administered intravenously.

An appropriate carrier for administering the cells may be selected byone of skill in the art by routine techniques. For example, thepharmaceutical carrier can be a buffered saline solution, e.g., cellculture media, and can include DMSO for preserving cell viability.

The quantity of APC appropriate for administration to a patient as acancer vaccine to effect the methods of the present invention and themost convenient route of such administration may be based upon a varietyof factors, as may the formulation of the vaccine itself. Some of thesefactors include the physical characteristics of the patient (e.g., age,weight, and sex), the physical characteristics of the tumor (e.g.,location, size, rate of growth, and accessibility), and the extent towhich other therapeutic methodologies (e.g., chemotherapy, and beamradiation therapy) are being implemented in connection with an overalltreatment regimen. Notwithstanding the variety of factors one shouldconsider in implementing the methods of the present invention to treat adisease condition, a mammal can be administered with from about 10⁵ toabout 10⁸ APC (e.g., 10⁷ APC) in from about 0.05 mL to about 2 mLsolution (e.g., saline) in a single administration. Additionaladministrations can be carried out, depending upon the above-describedand other factors, such as the severity of tumor pathology. In oneembodiment, from about one to about five administrations of about 10⁶APC is performed at two-week intervals.

DC vaccination can be accompanied by other treatments. For example, apatient receiving DC vaccination may also be receiving chemotherapy,radiation, and/or surgical therapy concurrently. Methods of treatingcancer using DC vaccination in conjunction with chemotherapy aredescribed in Wheeler et al., US Pat. Pub. No. 2007/0020297. In someembodiments, a patient receiving DC vaccination has already receivedchemotherapy, radiation, and/or surgical treatment for the cancer. Inone embodiment, a patient receiving DC vaccination is treated with aCOX-2 inhibitor, as described in Yu and Akasaki, WO 2005/037995.

Immunological Testing

The antigen-specific cellular immune responses of vaccinated subjectscan be monitored by a number of different assays, such as tetramerassays, ELISPOT, and quantitative PCR. The following sections provideexamples of protocols for detecting responses with these techniques.Additional methods and protocols are available. See e.g., CurrentProtocols in Immunology, Coligan, J. et al., Eds., (John Wiley & Sons,Inc.; New York, N.Y.).

Tetramer Assay

Tetramers comprised of recombinant MHC molecules complexed with peptidecan be used to identify populations of antigen-specific T cells. Todetect T cells specific for antigens such as HER-2, gp100 and MAGE-1,fluorochrome labeled specific peptide tetramer complexes (e.g.,phycoerythrin (PE)-tHLA) containing peptides from these antigens aresynthesized and provided by Beckman Coulter (San Diego, Calif.).Specific CTL clone CD8 cells are resuspended at 10⁵ cells/50 μl FACSbuffer (phosphate buffer plus 1% inactivated FCS buffer). Cells areincubated with 1 μl tHLA for 30 minutes at room temperature andincubation is continued for 30 minutes at 4° C. with 10 μl anti-CD8 mAb(Becton Dickinson, San Jose, Calif.). Cells are washed twice in 2 mlcold FACS buffer before analysis by FACS (Becton Dickinson).

ELISPOT Assay

ELISPOT assays can be used to detect cytokine secreting cells, e.g., todetermine whether cells in a vaccinated patient secrete cytokine inresponse to antigen, thereby demonstrating whether antigen-specificresponses have been elicited. ELISPOT assay kits are supplied from R & DSystems (Minneapolis, Minn.) and performed as described by themanufacturer's instructions. Responder (R) 1×10⁵ patients' PBMC cellsfrom before and after vaccination are plated in 96-well plates withnitrocellulose membrane inserts coated with capture Ab. Stimulator (S)cells (TAP-deficient T2 cells pulsed with antigen) are added at the R:Sratio of 1:1. After a 24-hour incubation, cells are removed by washingthe plates 4 times. The detection Ab is added to each well. The platesare incubated at 4° C. overnight and the washing steps will be repeated.After a 2-hour incubation with streptavidin-AP, the plates are washed.Aliquots (100 μl) of BCIP/NBT chromogen are added to each well todevelop the spots. The reaction is stopped after 60 min by washing withwater. The spots are scanned and counted with computer-assisted imageanalysis (Cellular Technology Ltd, Cleveland, Ohio). When experimentalvalues are significantly different from the mean number of spots againstnon-pulsed T2 cells (background values), as determined by a two-tailedWilcoxon rank sum test, the background values are subtracted from theexperimental values.

Quantitative PCR for IFN-γ Production

Quantitative PCR is another means for evaluating immune responses. Toexamine IFN-γ production in patients by quantitative PCR, cryopreservedPBMCs from patients' pre-vaccination and post-vaccinations samples andautologous dendritic cells are thawed in RPMI DC culture medium with 10%patient serum, washed and counted. PBMC are plated at 3×10⁶ PBMCs in 2ml of medium in 24-well plate; dendritic cells are plated at 1×10⁶/mland are pulsed 24 hour with 10 μg/ml tumor peptide in 2 ml in each wellin 24 well plate. Dendritic cells are collected, washed, and counted,and diluted to 1×10⁶/ml, and 3×10⁵ (i.e., 300 μl solution) added towells with PBMC (DC: PBMC=1:10). 2.3 μl IL-2 (300 IU/mL) is added every3-4 days, and the cells are harvested between day 10 and day 13 afterinitiation of the culture. The harvested cells are then stimulated withtumor cells or autologous PBMC pulsed with 10 μg/ml tumor peptide for 4hours at 37° C. On days 11-13, cultures are harvested, washed twice,then divided into four different wells, two wells using for control(without target); and another two wells CTL cocultured with tumor cells(1:1) if tumor cells are available. If tumor cells are not available, 10μg/ml tumor lysate is added to CTL. After 4 hours of stimulation, thecells are collected, RNA extracted, and IFN-γ and CD8 mRNA expressionevaluated with a thermocycler/fluorescence camera system. PCRamplification efficiency follows natural log progression, with linearregression analyses demonstrating correlation co-efficients in excess of0.99. Based on empirical analysis, a one-cycle difference is interpretedto be a two-fold difference in mRNA quantity, and CD8-normalized IFN-γquantities are determined. An increase of >1.5-fold in post-vaccinerelative to pre-vaccine IFN-γ is the established standard for positivetype I vaccine responsiveness.

In Vitro Induction of CTL in Patient-derived PBMCs

The following protocol can be used to produce antigen specific CTL invitro from patient derived PBMC. To generate dendritic cells, theplastic adherent cells from PBMCs are cultured in AIM-V mediumsupplemented with recombinant human GM-CSF and recombinant human IL-4 at37° C. in a humidified CO₂ (5%) incubator. Six days later, the immaturedendritic cells in the cultures are stimulated with recombinant humanTNF-α for maturation. Mature dendritic cells are then harvested on day8, resuspended in PBS at 1×10⁶ per mL with peptide (2 μg/mL), andincubated for 2 hours at 37° C. Autologous CD8+ T cells are enrichedfrom PBMCs using magnetic microbeads (Miltenyi Biotech, Auburn, Calif.).CD8+ T cells (2×10⁶ per well) are cocultured with 2×10⁵ per wellpeptide-pulsed dendritic cells in 2 mL/well of AIM-V medium supplementedwith 5% human AB serum and 10 units/mL rhIL-7 (Cell Sciences) in eachwell of 24-well tissue culture plates. About 20 U/ml of IL-2 is added 24h later at regular intervals, 2 days after each restimulation. On day 7,lymphocytes are restimulated with autologous dendritic cells pulsed withpeptide in AIM-V medium supplemented with 5% human AB serum, rhIL-2, andrhIL-7 (10 units/mL each). About 20 U/ml of IL-2 is added 24 h later atregular intervals, 2 days after each restimulation. On the seventh day,after the three rounds of restimulation, cells are harvested and testedthe activity of CTL. The stimulated CD8+ cultured cells (CTL) areco-cultured with T2 cells (a human TAP-deficient cell line) pulsed with2 μg/ml Her-2, gp100, AIM-2, MAGE-1, or IL13 receptor α2 peptides. After24 hours incubation, IFN-γ in the medium is measured by ELISA assay.

In Vivo Testing in Animal Models

Dendritic cell vaccination can be evaluated in animal models. Suitablemodels for brain cancers include injection models, in which cells of atumor cell line are injected into the animal, and genetic models, inwhich tumors arise during development.

To evaluate dendritic cell vaccination in an animal model, functionaldendritic cells are isolated from bone marrow derived cells of theanimal and differentiated in vitro in the presence of cytokines, asdetailed above. Mature dendritic cells are pulsed with tumor antigens(e.g., tumor antigens derived from the tumor cell line that will beimplanted into the animal, or synthetic peptides corresponding toepitopes of those antigens). Animals are implanted with cells of thetumor cell line. After implantation, animals are vaccinated withantigen-pulsed dendritic cells one or more times. Survival and immuneresponsiveness is measured.

Pharmaceutical Compositions

In various embodiments, the present invention provides pharmaceuticalcompositions including a pharmaceutically acceptable excipient alongwith a therapeutically effective amount of the inventive vaccinecomprising dendritic cells loaded with the antigens as described herein.“Pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic, and desirable, and includes excipients that are acceptablefor veterinary use as well as for human pharmaceutical use. Suchexcipients can be solid, liquid, semisolid, or, in the case of anaerosol composition, gaseous.

In various embodiments, the pharmaceutical compositions according to theinvention can be formulated for delivery via any route ofadministration. “Route of administration” can refer to anyadministration pathway known in the art, including, but not limited to,aerosol, nasal, transmucosal, transdermal or parenteral. “Parenteral”refers to a route of administration that is generally associated withinjection, including intraorbital, infusion, intraarterial,intracapsular, intracardiac, intradermal, intramuscular,intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal,intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous,transmucosal, or transtracheal. Via the parenteral route, thecompositions can be in the form of solutions or suspensions for infusionor for injection, or as lyophilized powders.

The pharmaceutical compositions according to the invention can alsocontain any pharmaceutically acceptable carrier. “Pharmaceuticallyacceptable carrier” as used herein refers to a pharmaceuticallyacceptable material, composition, or vehicle that is involved incarrying or transporting a compound of interest from one tissue, organ,or portion of the body to another tissue, organ, or portion of the body.For example, the carrier can be a liquid or solid filler, diluent,excipient, solvent, or encapsulating material, or a combination thereof.Each component of the carrier must be “pharmaceutically acceptable” inthat it must be compatible with the other ingredients of theformulation. It must also be suitable for use in contact with anytissues or organs with which it can come in contact, meaning that itmust not carry a risk of toxicity, irritation, allergic response,immunogenicity, or any other complication that excessively outweighs itstherapeutic benefits.

The pharmaceutical compositions according to the invention can bedelivered in a therapeutically effective amount. The precisetherapeutically effective amount is that amount of the composition thatwill yield the most effective results in terms of efficacy of treatmentin a given subject. This amount will vary depending upon a variety offactors, including but not limited to the characteristics of thetherapeutic compound (including activity, pharmacokinetics,pharmacodynamics, and bioavailability), the physiological condition ofthe subject (including age, sex, disease type and stage, generalphysical condition, responsiveness to a given dosage, and type ofmedication), the nature of the pharmaceutically acceptable carrier orcarriers in the formulation, and the route of administration. Oneskilled in the clinical and pharmacological arts will be able todetermine a therapeutically effective amount through routineexperimentation, for instance, by monitoring a subject's response toadministration of a compound and adjusting the dosage accordingly. Foradditional guidance, see Remington: The Science and Practice of Pharmacy(Gennaro ed. 21st edition, Williams & Wilkins PA, USA) (2005). In oneembodiment, a therapeutically effective amount of the vaccine cancomprise about 10⁷ tumor antigen-pulsed DC. In some embodiments, atherapeutically effective amount is an amount sufficient to reduce orhalt tumor growth, and/or to increase survival of a patient.

Kits

The present invention is also directed to kits to treat cancers (e.g.,neural cancers). The kits are useful for practicing the inventive methodof treating cancer with a vaccine comprising dendritic cells loaded withthe antigens as described herein. The kit is an assemblage of materialsor components, including at least one of the inventive compositions.Thus, in some embodiments, the kit includes a set of peptides forpreparing cells for vaccination. The kit can also include agents forpreparing cells (e.g., cytokines for inducing differentiation of DC invitro). The invention also provides kits containing a compositionincluding a vaccine comprising dendritic cells (e.g., cryopreserveddendritic cells) loaded with the antigens as described herein.

The exact nature of the components configured in the inventive kitdepends on its intended purpose. For example, some embodiments areconfigured for the purpose of treating neural cancers. Other embodimentsare configured for the purpose of treating brain tumors. In oneembodiment the brain tumor is a glioma. In another embodiment, the braintumor is GBM. In another embodiment, the brain tumor is an astrocytoma.In one embodiment, the kit is configured particularly for the purpose oftreating mammalian subjects. In another embodiment, the kit isconfigured particularly for the purpose of treating human subjects. Infurther embodiments, the kit is configured for veterinary applications,treating subjects such as, but not limited to, farm animals, domesticanimals, and laboratory animals.

Instructions for use can be included in the kit. “Instructions for use”typically include a tangible expression describing the technique to beemployed in using the components of the kit to effect a desired outcome,such as to treat cancer. For example, the instructions can compriseinstructions to administer a vaccine comprising dendritic cells loadedwith the antigens described herein to the patient. Instructions for usecan also comprise instructions for repeated administrations of thevaccine; for example, administering the three doses of the vaccine intwo week intervals.

Optionally, the kit also contains other useful components, such as,diluents, buffers, pharmaceutically acceptable carriers, syringes,catheters, applicators, pipetting or measuring tools, or other usefulparaphernalia as will be readily recognized by those of skill in theart.

The materials or components assembled in the kit can be provided to thepractitioner stored in any convenient and suitable ways that preservetheir operability and utility. For example the components can be indissolved, dehydrated, or lyophilized form; they can be provided atroom, refrigerated or frozen temperatures. The components are typicallycontained in suitable packaging material(s). As employed herein, thephrase “packaging material” refers to one or more physical structuresused to house the contents of the kit, such as inventive compositionsand the like. The packaging material is constructed by well knownmethods, preferably to provide a sterile, contaminant-free environment.The packaging materials employed in the kit are those customarilyutilized in cancer treatments or in vaccinations. As used herein, theterm “package” refers to a suitable solid matrix or material such asglass, plastic, paper, foil, and the like, capable of holding theindividual kit components. Thus, for example, a package can be a glassvial used to contain suitable quantities of an inventive compositioncontaining for example, a vaccine comprising dendritic cells loaded withthe antigens as described herein. The packaging material generally hasan external label which indicates the contents and/or purpose of the kitand/or its components.

EXAMPLES

The following examples are provided to better illustrate the claimedinvention and are not to be interpreted as limiting the scope of theinvention. To the extent that specific materials are mentioned, it ismerely for purposes of illustration and is not intended to limit theinvention. One skilled in the art can develop equivalent means orreactants without the exercise of inventive capacity and withoutdeparting from the scope of the invention.

Example 1 Human Studies

Phase I studies were initiated in two human patients to assess thesafety and efficacy of an immunotherapy trial using peripheral blooddendritic cells to present brain tumor-specific markers to the patient'simmune system. The details of these studies are described in Examples2-8 below.

No significant adverse events (grade III/IV toxicity) have been noted inthe patients thus far.

Example 2 Preparation of Autologous Dendritic Cells (DC)

Mononuclear cells were isolated from patients between days −28 to −14using leukapheresis. The COBE Spectra Apheresis System was used toharvest the mononuclear cell layer. Leukapheresis yields 10¹⁰ peripheralblood mononuclear cells (PBMC). These cells were allowed to becomeadherent for two hours at 37° C. in a tissue culture flask and washed inHBSS. Briefly, PBMC were seeded at a density of 1.4×10⁶ cells/cm² in185-cm² culture flasks (Nunc, Roskilde, Denmark) and allowed to adherefor 2 h at 37° C. Non-adherent cells were removed by washing four times.Adherent cells were cultured in RPMI 1640 supplemented with GM-CSF(Berlex) and IL-4 (R&D systems) for 5 days. On day 5, 50 ng/ml clinicalgrade TNF-α (R&D systems) was added to the culture medium for another3-4 days. On days 8-9, DCs were harvested and washed three times.

Patients underwent the following tests within 7 days of Leukapheresisprocedure: ABO/Rh; Antibody Screen; Syphilis; HBsAg; HBcAb; anti-HCV;anti-HIV1, 2; anti-HTLV I/II; and HIV-1/HCV by Nucleic Acid Testing(NAT) (ABO/Rh does not need to be repeated within 7 days ofLeukapheresis). Table 3 lists the names, manufacturers, methods, and anexplanation of each of these tests.

TABLE 3 TEST NAME REAGENT ABBREV. MANUFACTURER TEST METHOD BRIEFEXPLANATION Blood Group Gamma Biologicals, Olympus PK System Testing isnecessary to determine if red ABO/Rh Inc. & Ortho PK7200 an Automatedblood cells possess or lack A and/or B Diagnostic Systems,Pretransfusion Testing and D blood group antigens. Inc. a Johnson &System & Manual Tube Agglutination is a positive test result JohnsonCompany Test indicating the presence of the corresponding antigen.Normal human red blood cells possessing antigens will agglutinate in thepresence of antibody directed toward the antigens. Blood Group MicroTyping Systems, A/B/D Monoclonal ABO/Rh Inc. Grouping Card (Alternate)A/B/D Reverse Monoclonal Grouping Card MTS Anti-IgG Card Antibody ScreenImmucor, Inc. Capture-R Ready A qualitative test for the detection of AbNorcross, GA Screen unexpected blood group antibodies. Used to detectunexpected antibodies in the serum or plasma from donors. AntibodyScreen Micro Typing Systems, MTS Anti-IgG Card Ab (Alternate) Inc.Hepatitis B Ortho-Clinical Antibody to Hepatitis B This enzymeimmunoassay (EIA) Surface Antigen Diagnostics, Inc. Surface Antigendetects the presence of hepatitis B HBsAg Raritan, NJ (MurineMonoclonal): surface antigen (HbsAg) in human Peroxidase Conjugate serumor plasma. ORTHO Antibody to HbsAg ELISA Test System 3 Human Bio-RadLaboratories, Human This enzyme-linked immunoassay ImmunodeficiencyRedmond, WA Immunodeficiency (EIA) allows simultaneous detection ofVirus Types 1 and Virus Types 1 and 2 antibodies to HIV-1 and HIV-2. Itdoes 2 (Synthetic Peptide): not discriminate between HIV-1 and HIV-1/2Genetic Systems HIV- HIV-2 reactivity. 1/HIV-2 Peptide EIA SyphilisSerology Olympus America Inc., Olympus PK TP System This test isintended for the qualitative SYP Melville, NY detection of Treponemapallidum antibodies in human serum or plasma. This agglutination testutilizes fixed chicken erythrocytes sensitized with components of thepathogenic T. pallidum to detect antibodies in specimens. SyphilisSerology Immucor, Inc., Capture-S (Alternate) Norcross, GA Human T-BIOMERIEUX, Inc., Human T- This enzyme-linked immunoassay LymphotrophicDurham, NC Lymphotrophic Virus (EIA) detects antibodies to HTLV-I VirusType I and Type I and Type II and antibodies to HTLV-II. Type II(HTLV-I/II): HTLV-I/II Vironostika HTLV-I/II Microelisa System HepatitisC Virus ORTHO Clinical Hepatitis C Virus This enzyme immunoassayutilizes Encoded Antigen Diagnostics, Inc., Encoded Antigen recombinantantigens to detect antibody HCV Raritan, NJ (Recombinant c22-3, toHepatitis C virus (HCV). Presence of C200 and NS5) this antibodyindicates past or present ORTHO HCV Version HCV infection, or possibly acarrier 3.0 ELISA Test System state, but does not substantiateinfectivity nor immunity. The anti-HCV EIA 3.0 version test includesNS5, c200, and c22-3 recombinant antigens. The NS antigen is derivedfrom the polymerase of the HCV genome and allows antibody detection of agreater number of HCV epitopes. Nucleic Acid Gen-Probe ProcleixHIV-1/HCV This assay utilizes target amplification Testing (NAT)Incorporation, San Assay nucleic acid probe technology for the ProcleixDiego, CA detection of HIV-1 and/or HCV RNA. HIV-1/HCV RNA The screenassay is referred to as NAT “multiplex testing” which does notdiscriminate between HIV-1 and HCV RNA. Specimens found to be reactiveupon multiplex testing are then tested in HIV-1 and HCV DiscriminatoryAssays (dHIV and dHCV assays) to determine if they are reactive for HIV,HCV, both or neither. All assays have a chemiluminescent signal producedby a hybridized probe, which is measured by a luminometer and reportedas Relative Light Units (RLU).

Example 3 Preparation of Vaccines

Human leukocyte antigen A1 (HLA-A1, or A1) and human leukocyte antigenA2 (HLA-A2, or A2) positive patients with recurrent brain stem glioma orglioblastoma were identified. Dendritic cells, prepared as described inExample 2, were pulsed with peptide epitopes of tumor antigens that bindto HLA-A1 or HLA-A2, to load the cells with the antigens, prior tofrozen storage. The peptide epitopes were from the following tumorantigens: MAGE-1, HER-2, AIM-2, TRP-2, gp100, and interleukin-13receptor α2. The sequences of these peptide epitopes used in thesestudies are listed in Table 4, below. Other epitopes for these antigenscan also be used.

TABLE 4 Tumor Antigen Peptides Antigen HLA-A1 epitope Antigen HLA-A2epitope AIM-2 RSDSGQQARY TRP-2 SVYDFFVWL (SEQ ID NO: 3) (SEQ ID NO: 5)MAGE-1 EADPTGHSY GP100 ITDQVPFSV (SEQ ID NO: 4) (SEQ ID NO: 6) HER-2KIFGSLAFL (SEQ ID NO: 7) IL-13R α2 WLPFGFILI (SEQ ID NO: 8)

Tumor antigen epitopes were purchased from Clinalfa (Läufelfingen,Switzerland).

On the day prior to immunization, days 8-9 DC cultures were washed threetimes in dPBS, resuspended at 10⁶ cells/ml in complete media and thencoincubated with tumor associated antigen peptides (10 μg/ml perantigen, reconstituted in 10% DMSO). The dendritic cells were incubatedwith the peptides at 37°/5% CO₂ for 16-20 hours on a tissue rotator tofacilitate interaction.

Mature (d8-9) DC were frozen as follows: DC are resuspended in cryotubes at various concentrations (1×10⁷ cells per ml in autologousfreezing medium (10% DMSO and 90% autologous serum), then immediatelytransferred to 1.8 ml cryo tubes (cryo tube vials, Nunc, Brand Products,Roskilde, Denmark), slowly frozen to −80° C. by using a cryo freezingcontainer (Nalgene cryo 1° C. freezing container, rate of cooling −1°C./min (Fisher Scientific, CA)) and finally transferred into the gasphase of liquid nitrogen until use.

Sterility testing was conducted to confirm suitability for use. To teststerility, APC were cultured in RPMI medium, 10% heat-inactivated humanAB serum, and 1% Gentamicin “GIBCO”. Purchased Human AB serum was heatinactivated to 56° C. for one hour prior to preparation of completemedium. Each batch of complete medium was prepared on the day of eachblood draw and sterile filtered (0.22 μm filter; Nalgene) prior to use.Complete media was refrigerated during the 9 day APC culture period. Onday 2 of the APC culture, an aliquot of spent culture media was removedand subjected to sterility testing using BacT/Alert system with aerobicand anaerobic bottles that are cultured for 14 days total in anautomated system.

In addition, a gram stain, sterile culture, mycoplasma, and LALendotoxin assays are performed on the final product before theadministration to the patient.

Acceptance Criteria for Test Article: 5 Eu/ml/kg of patient (endotoxin);no growth in sterility cultures; no bacteria seen by gram staining,hybridization control positive, water control negative, and the vaccineproduct exhibiting no sustained logarithmic increase in fluorescenceintensity for mycoplasma QPCR.

Example 4 Protocol for Administering the Vaccine

For immunization, the patient received 107 tumor antigen-pulseddendritic cells, intradermally in 1 ml autologous freezing media in theaxillary region. The patient was monitored for two hourspost-immunization. Patients can receive pretreatment with 50 mgdiphenhydramine and 650 mg of Tylenol, both orally (only as needed totreat symptoms or for the prevention of the recurrence of any priorstudy-agent-associated symptoms). The schedule of vaccineadministration, and pre- and post-vaccine testing is shown in Table 5.The schedule of blood draws for vaccine preparation and testing is shownin Table 6.

TABLE 5 Vaccination and Immunological Testing Schedule Day Events −28 to−14 A patient is screened and informed consent is obtained. MRI, Blooddraw for serum (to supplement freezing medium), Immunological tests(pretreatment), Leukaphe- resis and preparation of dendritic cells isperformed. 0 TAA-pulsed APC vaccination (1st). 14 TAA -pulsed APCvaccination (#2). 28 TAA-pulsed APC vaccination (#3). 56 Immunologicaltests, MRI, AE assessment, blood tests, targeted exam, Karnofsky (week10), MRI every 2 months, are performed. 180 Immunological tests areperformed (month 4).

TABLE 6 Specific Blood Draw and Volume Schedule Day Event Vol. −28 Blooddraw for 100 ml  serum + DC's −28 Immunological 70 ml tests 56Immunological 70 ml tests 180 Immunological 70 ml tests

Example 5 Screening and Baseline Evaluations

The following clinical and laboratory evaluations occur within days −28to −9 unless otherwise noted.

-   -   Objective Signs and Symptoms: Includes vital signs (blood        pressure, pulse, temperature and respirations), and weight.        (Screening and repeat Day 0.)    -   History and Review of Systems: Screening and Review of Systems        on Day −28 to −9, repeated Neurological exam on Day 0.    -   Karnofsky Performance Status (Screening)

Karnofsky Index:

100 Normal; no complaints; no evidence of disease. 90 Able to carry onnormal activity; minor signs or symptoms. 80 Normal activity witheffort; some signs or symptoms. 70 Cares for self; unable to carry onnormal activity or to do active work. 60 Requires occasional assistance;able to care for most needs. 50 Requires considerable assistance; ableto care for most needs. 40 Disabled; requires special care andassistance. 30 Severely disabled; hospitalization necessary; activesupportive treatment necessary. 20 Very sick; hospitalization necessary;active supportive treatment necessary. 10 Moribund; rapidly progressingfatal process. 0 Dead.

-   -   MRI of Brain with and without contrast    -   Urinalysis: Normal routine urinalysis    -   Serum Chemistries: Includes uric acid, calcium, phosphorous,        magnesium, amylase, triglycerides, transaminases (AST, ALT),        alkaline phosphatase, LDH, total bilirubin, BUN, creatinine,        albumin, total protein, electrolytes, glucose (Screening), ANA,        and TSH.    -   Hematology: Complete blood count (CBC), differential, platelets,        and coagulation tests should include PT (Prothrombin Time) and        PTT (Partial Thromboplastin Time). PT and PTT will be done at        screening only and are repeated if clinically indicated.

Example 6 Interval Evaluations

Objective Signs and Symptoms: vital signs (blood pressure, pulse,temperature and respiration) and weight will be done.

Review of Systems: Neurological exam will be done on Study Days (i.e.,days when the patient sees a physician).

Karnofsky Performance Status are done on Study Days.

Serum Chemistries: Include uric acid, calcium phosphorous, magnesium,amylase, triglycerides, transaminases (AST, ALT), alkaline phosphatase,LDH, total bilirubin, BUN, creatinine, albumin, total protein,electrolytes glucose, ANA, and TSH.

Hematology: Complete blood count (CBC), differential platelets andcoagulation tests should include PT (Prothrombin time) and PTT (PartialThromboplastin time).

MRI of brain with and without contrast (q2 months).

Example 7 Vaccination Modification and General Management of Toxicities

A Table for Grading Severity of Adverse Experience (AE) is used toachieve consistency in response to drug/treatment toxicities. Toxicitiesare graded on the NIH Common Toxicity Criteria, a 1-4 basis scale. If atoxicity is experienced, the treatment level or dose is modified (ifapplicable) as outlined below according to the grade of toxicityobserved. AEs related to neurological deficits or post-vaccinationtherapy due to tumor progression. All SAEs will be reported untilsurvival.

For any Grade 1 toxicity there will be no dose modification.

If a Grade 2 toxicity develops, the patient will not receive a plannedsubsequent vaccine injection until values return to Grade 1 or less forat least one week.

All vaccine administrations will cease for any patient who experiencesany of the following outcomes within one month following any vaccineinjection: any grade 3 or 4 adverse event; a grade 2 (or greater)allergic adverse event; or a grade 2 (or greater) neurologic adverseevent not readily attributable to the tumor.

Symptomatic therapy such as analgesics or other helpful therapy can beadministered if deemed necessary.

Example 8 Primary Safety and Efficacy Analyses

The primary safety outcome is number of Grade 3 or 4 toxicities. Safetyoutcomes are followed over a period of one year following the last studyagent dose administration.

The primary endpoint is survival time (from date of vaccination to dateof death or the last date known alive if death was not observed).

The secondary endpoints of progression free survival after vaccinationare measured radiologically with MRI scan of the brain with and withoutgadolinium. Patients will undergo an MRI every two months after the laststudy agent administration Standardized response criteria as outlinedbelow have been adopted.

Complete Response: Complete disappearance of all tumor on MRI with astable or improving neurologic examination.

Partial Response: Greater than or equal to 50% reduction in tumor sizeon volumetric MRI scan with a stable or improving neurologicexamination.

Progressive Disease or Recurrent Disease: Progressive neurologicabnormalities or a new or greater than 25% increase in the volume of thegadolinium-enhancing tumor by MRI scan.

Stable Disease: A patient whose clinical status and MRI volumetrics donot meet the criteria for partial response or progressive disease.

Dose limiting toxicities are followed for one month after the last studyagent administration.

Example 9 In Vivo Testing in an Animal Model

The following in vivo animal experiments demonstrate the efficacy of adendritic cell vaccine. To isolate functional dendritic cells (DC),cells were harvested from rat bone marrow. Bone marrow suspensions weresupplemented with GM-CSF (50 ng/ml) and IL-4 (100 ng/ml) for 8 days,which have been shown to induce the differentiation of functionaldendritic cells. Mature dendritic cells from culture were positivelyidentified based on their surface antigen expression via flow cytometry(FACS). Dendritic cells were positively identified based on theirexpression of MHC Class II, MHC Class I, CD11b/c, and Thy1.1, and theirlack of CD3 and CD8 expression. Cultures enriched for dendritic cellswere pulsed (co-cultured) overnight with acid eluted tumor peptides fromsyngeneic 9 L rat glioma cells.

In these animal experiments, 9 L glioma cells were stereotacticallyimplanted into the right cerebral hemisphere of Fischer 344 rats. Oneweek after tumor implantation, animals were injected subcutaneously with5×10⁵ 9 L peptide-pulsed dendritic cells, unpulsed dendritic cells orcontrol media. Three weekly injections were given. Animals in each ofthe treatment groups were followed for survival. The results revealedthat a significantly higher percentage of animals treated with 9 Lpeptide-pulsed dendritic cells were still surviving at 20 days(10/12=83%) compared to those treated with unpulsed cells (2/6=33%) oruntreated animals (3/10=30%).

Example 10 Enhanced Immune Responsiveness to T cell Epitopes withDibasic Motifs

In studies using mouse lysozyme-M (ML-M) as a model self Ag, it wasobserved that mice of diverse MHC haplotypes were tolerant to native(unmutated) ML-M and peptide forms of certain T cell epitopes. It washypothesized that tolerance to a given epitope was not an inherentstructural characteristic, but was attributable in part to inefficientprocessing of the epitope, owing either to the absence of a proteolyticcleavage site adjacent to that determinant, or to the inaccessibility tothe proteolytic enzyme(s) of an existing cleavage site within thatregion of the molecule. In either case, the provision of a newproteolytic cleavage site adjacent to a cryptic determinant may permitscission of the peptide at that site, making the previously crypticepitope region available for binding to the appropriate MHC molecule,and lead to presentation to specific T cells of that determinant as aneodominant epitope on the APC surface. To test this proposition, adibasic motif, consisting of two contiguous basic amino acid residues,e.g., arg-arg (RR) or arg-lys (RK), was used.

The targeted regions within ML-M included residues 19-31, which containcryptic epitopes for mice of the H-2^(k) haplotype, shown in Table 7.

TABLE 7 Creation of Dibasic Motifs in Flanking Region of a DefinedCryptic Epitope Within ML-M ML-M Target Dominant Epitope for DibasicResulting Epitope^(a) Mouse Strain (H-2)^(a) ML-M T cell epitope SiteDibasic Motif 19-30 C3H/HeJ (H-2^(k)) RR-GYYGVSLADWVC 18 RR (SEQ ID NO:60) 19-30 C3H/HeJ (H-2^(k)) GYYGVSLADWVC-RR 31 RR (SEQ ID NO: 61) 19-30C3H/HeJ (H-2^(k)) RR-GYYGVSLADWVC-RR 18 + 31 RR-RR (SEQ ID NO: 62)

Groups of mice (3 per group) were immunized by IV injection of eachpeptide (ML-M, p19-30, or 18R31R. T cells were isolated from lymph nodeof the animals. Single cells in 96 well plates were recalled with eachof the peptides and response to each of the peptides was measured.Interestingly, C3H mice challenged with ML-M failed to respond to theimmunogen and to various peptides of this self lysozyme, includingpeptide 19-31, whereas immunization with RR-p18-31-RR raised a potent Tcell response to this altered lysozyme as well as to p19-30 (FIG. 1).These results demonstrate that the dibasic site RR/KK mediated anefficient processing of the epitope 19-30, leading to activation ofspecific T cells. Furthermore, the T cells primed by RR-p18-31-RR couldbe restimulated in vitro with RR-p18-31—RR, but not by ML-M or peptidesfrom other regions of ML-M. These results further confirmed theefficient presentation by the APC of epitope 19-30, but not ML-M tospecific T cells, and also demonstrated the T cell cross-reactivity withsynthetic p19-30.

Studies related to these studies with ML-M are performed with HER-2.HER-2/neu is a self-antigen expressed by tumors and nonmalignantepithelial tissues. The possibility of self-tolerance toHER-2/neu-derived epitopes has raised questions concerning their utilityin antitumor immunotherapy. Altered HER-2/neu peptide ligands capable ofeliciting enhanced immunity to tumor-associated HER-2/neu epitopes maycircumvent this problem. The human CTL peptide HER-2/neu (435-443)modified with RR or RK dibasic motifs is an example of an alteredpeptide ligand of HER2.

The following exemplary dibasic-modified forms of HER2 peptides areobtained from Macromolecular Resources and Global Peptide Services:hHER-2(9₄₃₅) (RRILHNGAYSLRR) (SEQ ID NO:1) and RRKIFGSLAFLRR (SEQ IDNO:2).

Murine In Vivo Lymphocyte Proliferation Assay

To test immunogenicity in an animal model, mice are immunized s.c.either altered hHER-2 peptide or with a peptide of hHER-2 (1 mg/mleach), each emulsified in complete Freunds adjuvant (CFA; InvitrogenLife Technologies) (1:1, v/v). After 8 or 9 days, the draining lymphnode cells (5×10⁵/well) of these mice are tested in a proliferationassays using the appropriate peptides. Purified protein derivative (PPD)(Mycos Research) is used as a positive control. The incorporation ofradioactivity ([³H]thymidine) is assayed by liquid scintillationcounting. The results will be expressed either as counts per minute(cpm) or as a stimulation index (stimulation index=cpm with recallantigen/cpm with cells in medium alone).

In Vitro Cell Proliferation Assay Using Paraformaldehyde (PF)—Fixed APC

Cell proliferation is tested in vitro using the following assay.Briefly, DC from PBMC are used as APC. APC are fixed by incubation with0.5% paraformaldehyde (PF; Sigma-Aldrich) for 10 min at room temperatureeither before or after pulsing with antigen. Naive unfixed APC are usedas a control for fixed APC. Antigen-primed T cells are purified from LNCand spleen of antigen-challenged mice using a nylon wool column(Polysciences), and then cultured (1.5×10⁵/well) with fixed/unfixed APC(3.75×10⁵/well). APC plus T cells without Ag, and T cells with Ag only(no APC) serve as additional controls. The results are expressed as cpmor stimulation index, as described above.

T Cell ATP Release Function Assay

DC are pulsed with peptides for 16-20 hours, then incubated with CD4/CD8T cells overnight and analyzed with an ATP releasing T cellproliferation assay. Table 8 lists the correlation of ATP levels withimmune responsiveness, and suitability for a vaccine.

TABLE 8 Correlation of ATP Level with Immune Response ATP RangeInterpretation (ng/mL) Immune Response Vaccine 225 Low − 226-524moderate +/− 525 strong +

Measurement of the Cytokine Levels

LNC of antigen-primed mice are restimulated with antigen in vitro for 48h. Thereafter, the culture supernatants are collected and assayed byELISA using kits for IFN-γ and IL-4 (BioSource International). Theabsorbance is read at 450 nm using MicroElisa autoreader (MolecularDevices). The results are expressed as Δpg/ml (=cytokine secreted by LNCwith Ag—cytokine in medium control). The Th1/Th2 ratio is derived fromthe levels of IFN-γ/IL-4, respectively.

Determination of the Serum Levels of Ag-Specific Abs

The level of antibodies (total IgG, IgG1, and IgG2a) in sera is testedat different dilutions and detected by ELISA using different antigen(0.1 μg/well of a high binding ELISA plate (Greiner Bioscience)) and theappropriate HRP-conjugated secondary Ab against total Ig, or Ab specificfor the IgG1 or IgG2a isotype (BD Pharmingen) (1:1000) followingstandard procedures. The results will be expressed as OD (450 nm) units.

Example 11 IFN-γ Production of Altered Peptide ligands (APL)-SpecificCTLs

The immunogenicity of the APLs (also referred to herein as superagonistepitopes) in two HLA-A*0201 GBM patients were tested to determine thecapacity of prime CTL responses in vitro. The results of theseexperiments are depicted in FIGS. 2 and 3.

For the gp100 APLs, among six different CTLs stimulated by six differentpeptides, No. 38 showed the highest IFN-γ level when targeting T2 pulsedwith gp100 native peptide in both patients. There was a significantdifference (P<0.05) when CTL No. 38 was compared with CTL generated bythe gp100 (2M) peptide.

For the Her-2 APLs, among four different CTLs stimulated by fourdifferent peptides, No. 19 showed the highest IFN-γ level when targetingT2 pulsed Her-2 native peptide in both patients. There was a significantdifference (P<0.05) when CTL No. 19 was compared with CTL generated byCTL No. 52 and CTL generated by Her-2 native peptide. In conclusion,among the tested peptides, No. 38 and No. 19 are the best superagonists.

Tables 9 and 10 list the altered peptide ligand (i.e., superagonistepitope) sequences of gp-100 and Her-2, respectively. The bolded lettersindicate the amino acid that is substituted in place of the native aminoacid residue. gp100 (2M) is an analog of the native gp100 with areplacement of the amino acid T with M. The native gp100 peptidesequence is as follows: ITDQVPFSV (SEQ ID NO:6). Peptide No. 52 is anative HER-2 peptide and not an altered peptide. The peptides weredissolved in 5% DMSO at 2 mg/ml and stored at −20° C. until taken outfor use.

TABLE 9 Amino acid sequences of gp100 APL gp100 (2M) H— I T D Q V P F SV —NH₂ SEQ ID NO: 6 No. 8 H— F L D Q V P Y S V —NH₂ SEQ ID NO: 63 No. 22H— F M D Q V P Y S V —NH₂ SEQ ID NO: 64 No. 38 H— Y M D Q V P Y S V —NH₂SEQ ID NO: 65 No. 62 H— I L D Q V P F S V —NH₂ SEQ ID NO: 66 No. 63 H— IM D Q V P F S V —NH₂ SEQ ID NO: 67

TABLE 10 Amino acid sequences of HER-2 APL No. H— F M A N V A I P H L—NH₂ SEQ ID NO: 68 cp1 19 No. H— F M H N V P I P Y L —NH₂ SEQ ID NO: 69cp14 32 No. H— F Y A N V P S P H L —NH₂ SEQ ID NO: 70 cp23 41 No. H— V MA G V G S P Y V —NH₂ SEQ ID NO: 71 native, 52 C- terminal amide

Example 12 In Vitro Induction of CTL in Patient-Derived PBMCs andStimulation with Altered Peptide Ligands Superagonist Peptides

The following assays were used to further evaluate immune responses tothe altered peptide ligands described in Example 11. To generatedendritic cells, plastic-adherent cells from human PBMCs were culturedin AIM-V medium supplemented with recombinant human GM-CSF andrecombinant human IL-4 at 37° C. in a humidified CO₂ (5%) incubator. Sixdays later, the immature dendritic cells were stimulated withrecombinant human TNF-α for maturation. Mature dendritic cells were thenharvested on day 8, resuspended in PBS at 1×10⁶ per mL with peptide (2μg/mL), and incubated for 2 hours at 37° C.

Autologous CD8+ T cells were enriched from PBMCs using magneticmicrobeads (Miltenyi Biotech, Auburn, Calif.). CD8+ T cells (2×10⁶ perwell) were cocultured with 2×10⁵ per well peptide-pulsed dendritic cellsin 2 mL/well of AIM-V medium supplemented with 5% human AB serum and 10units/mL rhIL-7 (Cell Sciences) in each well of 24-well tissue cultureplates. The gp100 and Her-2 peptides used for pulsing the dendriticcells are described in Example 11. On the next day and then every 3days, 300 IU/ml IL-2 was added to the medium. On day 7, lymphocytes wererestimulated with autologous dendritic cells pulsed with peptide inAIM-V medium supplemented with 5% human AB serum, rhIL-2, and rhIL-7 (10units/mL each).

CTL Co-Culture with GBM Tumor Cells

After three cycles of stimulation, on day 20, the CD8+ cultured cells(CTL) were co-cultured with four GBM cell lines, which are both HLA-A2and HER-2, gp 100 positive cell lines. After a 24 hour incubation, IFN-γin the medium was measured by ELISA assay. The data are shown in FIG. 4and FIG. 6.

ELISPOT Assays

ELISPOT assays were performed with kits (R & D Systems, Minneapolis,Minn.) according to the manufacturer's instructions. After three cyclesof stimulation, on day 20, the CD8+ cultured cells (CTL) were plated in96-well plates with nitrocellulose membrane inserts coated with captureantibody (Ab). Target cells (T2 pulsed HER-2 native peptide for FIG. 5or T2 pulsed gp100 native peptide for FIG. 7) were added at theCTL:target ratio of 1:1. After a 24 hour incubation, cells were removedby washing the plates 4 times. The detection Ab was added to each well.The plates were incubated at 4° C. overnight and the washing steps wererepeated. After a 2 hour incubation with streptavidin-AP, the plateswere washed. Aliquots (100 μl) of BCIP/NBT chromogen were added to eachwell to develop the spots. The reaction was stopped after 60 minutes bywashing with water. The spots were scanned and counted withcomputer-assisted image analysis (Cellular Technology Ltd, Cleveland,Ohio). When experimental values were significantly different from themean number of spots against non-pulsed T2 cells (background values), asdetermined by a two-tailed Wilcoxon rank sum test, the background valueswere subtracted from the experimental values. The coefficient ofvariation of intra-assay for ELISPOT in these experiments was less than10%. The data are shown in FIG. 5 and FIG. 7.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method for treating a cancer in a patient, the method comprising:administering to the patient a composition comprising dendritic cells,wherein the dendritic cells present on their surface peptide epitopescomprising amino acid sequences corresponding to epitopes of at leastfour of the following six antigens: tyrosinase-related protein (TRP)-2,Melanoma-associated Antigen-1 (MAGE-1), HER-2, interleukin-13 receptorα2 (IL-13 receptor α2), gp100, and Antigen isolated from ImmunoselectedMelanoma-2 (AIM-2), wherein at least one of the peptide epitopes is asuperagonist peptide epitope.
 2. The method of claim 1, wherein thedendritic cells acquired the peptide epitopes in vitro by exposure tosynthetic peptides comprising the peptide epitopes.
 3. (canceled)
 4. Themethod of claim 1, wherein the cancer is a glioma.
 5. The method ofclaim 2, wherein the synthetic peptides comprise 9-13 amino acidresidues.
 6. The method of claim 1, wherein the dendritic cells comprisepeptide epitopes corresponding to TRP-2, MAGE-1, HER-2, IL-13 receptorα2, gp100, and AIM-2.
 7. The method of claim 1, wherein the compositionis administered to the patient two or more times.
 8. The method of claim1, wherein the composition comprises between 10⁵ to 10⁷ dendritic cells.9. The method of claim 1, wherein the peptide epitopes comprise at leastone peptide with one of the following sequences: RSDSGQQARY (SEQ ID NO:3) from AIM-2; EADPTGHSY (SEQ ID NO: 4) from MAGE-1; SVYDFFVWL (SEQ IDNO: 5) from TRP-2; ITDQVPFSV (SEQ ID NO: 6) from gp100; KIFGSLAFL (SEQID NO: 7) from HER-2; and WLPFGFILI (SEQ ID NO: 8) from IL-13 receptorα2.


10. The method of claim 1, wherein the peptide epitopes comprise one orboth of the following superagonist peptide sequences: YMDQVPYSV (SEQ IDNO: 65) from gp100; or FMANVAIPHL (SEQ ID NO: 68) from HER-2.


11. The method of claim 1, wherein the peptide epitopes comprise one ofthe following superagonist peptide sequences: FLDQVPYSV (SEQ ID NO: 63)from gp100 ILDQVPFSV (SEQ ID NO: 66) from gp100 IMDQVPFSV (SEQ ID NO:67) from gp100.


12. The method of claim 1, wherein the peptide epitopes comprise one ofthe following superagonist peptide sequences: FMHNVPIPYL (SEQ ID NO: 69)from HER-2; or FYANVPSPHL (SEQ ID NO: 70) from HER-2.


13. The method of claim 1, wherein the composition comprises autologousdendritic cells.
 14. The method of claim 4, wherein the glioma isglioblastoma multiforme.
 15. The method of claim 4, wherein the gliomais an astrocytoma.
 16. A method for preparing a cell vaccine fortreating a glioma, the method comprising: obtaining bone marrow derivedmononuclear cells from a patient, culturing the mononuclear cells invitro under conditions in which mononuclear cells become adherent to aculture vessel, selecting a subset of the mononuclear cells comprisingadherent cells, culturing the adherent cells in the presence of one ormore cytokines under conditions in which the cells differentiate intoantigen presenting cells, culturing the antigen presenting cells in thepresence of synthetic peptides, the peptides comprising amino acidsequences corresponding to epitopes of at least four of the followingsix antigens: TRP-2, MAGE-1, HER-2, IL-13 receptor α2, gp100, and AIM2,wherein at least one of the peptide epitopes is a superagonist peptideepitope, under conditions in which the cells present the peptides onmajor histocompatibility class I molecules, thereby preparing a cellvaccine.
 17. The method of claim 14, wherein the synthetic peptidescomprise epitopes corresponding to TRP-2, MAGE-1, HER-2, IL-13 receptorα2, gp100, and AIM-2.
 18. (canceled)
 19. The method of claims 16,wherein the one or more cytokines comprise granulocyte macrophage colonystimulating factor and interleukin-4 (IL-4).
 20. The method of claim 16,wherein the one or more cytokines comprise tumor necrosis factor-α(TNF-α).
 21. The method of claim 16, wherein the bone marrow derivedcells are obtained from a patient with a glioma, and wherein the cellvaccine is prepared to treat the patient.
 22. The method of claim 16,wherein the synthetic peptides comprise at least one peptide comprisingone of the following sequences: RSDSGQQARY (SEQ ID NO: 3) from AIM-2;EADPTGHSY (SEQ ID NO: 4) from MAGE-1; SVYDFFVWL (SEQ ID NO: 5) fromTRP-2; ITDQVPFSV (SEQ ID NO: 6) from gp100; KIFGSLAFL (SEQ ID NO: 7)from HER-2; and WLPFGFILI (SEQ ID NO: 8) from IL-13 receptor α2.


23. The method of claim 22, wherein the synthetic peptides comprise oneor both of the following superagonist peptide sequences: YMDQVPYSV (SEQID NO: 65) from gp100; or FMANVAIPHL (SEQ ID NO: 68) from HER-2.


24. The method of claim 22, wherein the peptide epitopes comprise one ofthe following superagonist peptide sequences: FLDQVPYSV (SEQ ID NO: 63)from gp100 ILDQVPFSV (SEQ ID NO: 66) from gp100 IMDQVPFSV (SEQ ID NO:67) from gp100.


25. The method of claim 22, wherein the peptide epitopes comprise one ofthe following superagonist peptide sequences: FMHNVPIPYL (SEQ ID NO: 69)from HER-2; or FYANVPSPHL (SEQ ID NO: 70) from HER-2.


26. A kit for preparing a cell vaccine for treating a cancer, the kitcomprising: a set of synthetic peptides, the peptides comprising aminoacid sequences corresponding to epitopes of at least four of thefollowing six antigens: TRP-2, MAGE-1, HER-2, IL-13 receptor α2, gp100,and AIM2, wherein at least one of the peptides is a superagonistpeptide.
 27. (canceled)
 28. The kit of claim 26, wherein the syntheticpeptides comprise at least one of the following sequences: RSDSGQQARY(SEQ ID NO: 3) from AIM-2; EADPTGHSY (SEQ ID NO: 4) from MAGE-1;SVYDFFVWL (SEQ ID NO: 5) from TRP-2; ITDQVPFSV (SEQ ID NO: 6) fromgp100; KIFGSLAFL (SEQ ID NO: 7) from HER-2; and WLPFGFILI (SEQ ID NO: 8)from IL-13 receptor α2.


29. The kit of claim 26, wherein the synthetic peptides comprise one orboth of the following superagonist peptide sequences: YMDQVPYSV (SEQ IDNO: 65) from gp100; or FMANVAIPHL (SEQ ID NO: 68) from HER-2.


30. The kit of claim 26, wherein the synthetic peptides comprise one ofthe following superagonist peptide sequences: FLDQVPYSV (SEQ ID NO: 63)from gp100 ILDQVPFSV (SEQ ID NO: 66) from gp100 IMDQVPFSV (SEQ ID NO:67) from gp100.


31. The kit of claim 26, wherein the synthetic peptides comprise one ofthe following superagonist peptide sequences: FMHNVPIPYL (SEQ ID NO: 69)from HER-2; or FYANVPSPHL (SEQ ID NO: 70) from HER-2.


32. The kit of claim 26, wherein the kit further comprises cytokines forinducing differentiation of bone marrow derived cells into antigenpresenting cells in vitro.
 33. A composition comprising dendritic cells,wherein the dendritic cells comprise peptide sequences comprisingepitopes corresponding to epitopes of at least four of the following sixantigens: TRP-2, MAGE-1, HER-2, IL-13 receptor α2, gp100, and AIM2,wherein at least one of the peptide epitopes is a superagonist peptide,and wherein the dendritic cells acquired the peptide epitopes in vitroby exposure to synthetic peptides comprising the peptide epitopes.
 34. Acomposition comprising a peptide with one of the following sequences:FMANVAIPHL (SEQ ID NO: 68) from HER-2; FMHNVPIPYL (SEQ ID NO: 69) fromHER-2; or FYANVPSPHL (SEQ ID NO: 70) from HER-2.


35. The composition of claim 34, wherein the composition is present inan amount sufficient to induce an immune response.
 36. A compositioncomprising dendritic cells, wherein the dendritic cells comprise apeptide epitope with one of the following amino acid sequences:FMANVAIPHL (SEQ ID NO: 68) from HER-2; FMHNVPIPYL (SEQ ID NO: 69) fromHER-2; or FYANVPSPHL (SEQ ID NO: 70) from HER-2.


37. A method for treating a cancer in a patient, the method comprising:administering to the patient a composition comprising dendritic cells,wherein the dendritic cells comprise a superagonist peptide with one ofthe following sequences: FMANVAIPHL (SEQ ID NO: 68) from HER-2;FMHNVPIPYL (SEQ ID NO: 69) from HER-2; or FYANVPSPHL (SEQ ID NO: 70)from HER-2.